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. 2014 Aug 12;111(32):11679-84.
doi: 10.1073/pnas.1410900111. Epub 2014 Jul 28.

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

Matthew T Weinstock  1 , Michael T Jacobsen  1 , Michael S Kay  2
Affiliations

Synthesis and folding of a mirror-image enzyme reveals ambidextrous chaperone activity

Matthew T Weinstock et al. Proc Natl Acad Sci U S A. .

Abstract

Mirror-image proteins (composed of D-amino acids) are promising therapeutic agents and drug discovery tools, but as synthesis of larger D-proteins becomes feasible, a major anticipated challenge is the folding of these proteins into their active conformations. In vivo, many large and/or complex proteins require chaperones like GroEL/ES to prevent misfolding and produce functional protein. The ability of chaperones to fold D-proteins is unknown. Here we examine the ability of GroEL/ES to fold a synthetic d-protein. We report the total chemical synthesis of a 312-residue GroEL/ES-dependent protein, DapA, in both L- and D-chiralities, the longest fully synthetic proteins yet reported. Impressively, GroEL/ES folds both L- and D-DapA. This work extends the limits of chemical protein synthesis, reveals ambidextrous GroEL/ES folding activity, and provides a valuable tool to fold d-proteins for drug development and mirror-image synthetic biology applications.

Keywords: peptide synthesis; protein folding.

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Conflict of interest statement

Conflict of interest statement: M.S.K. is a Scientific Director, consultant, and equity holder of the d-Peptide Research Division of Navigen, which is commercializing d-peptide inhibitors of viral entry.

Figures

Fig. 1.
Fig. 1.
Total chemical synthesis of 312-residue DapA. (A) Target amino acid sequence, including N-terminal half (DapA 1–4, green), C-terminal half (DapA 5–8, blue), N-terminal His tag and thrombin cleavage site (italics), cysteines (red), ligation sites (bold and underline), and A77C mutation (red arrow). (B) Final synthetic strategy, including peptide segments (DapA 1–4, green and DapA 5–8, blue) with ligation hydrazide and cysteine residues indicated. Oxidation (hydrazide to azide), native chemical ligation (NCL), and acetamidomethyl (ACM) cysteine deprotection steps are also indicated.
Fig. 2.
Fig. 2.
Validation of the DapA A77C mutation. (A) Natural sequence diversity at position 77 from Protein BLAST analysis. (B) Structure of DapA tetramer (PDB ID code 1DHP) showing, on one subunit, the surface-exposed alanine at position 77 (cyan), natural cysteine residues (green), and catalytic lysine at position 181 in the active site (red). (C) Enzyme activity of recombinant native WT and A77C DapA. Error bars indicate SD of at least three measurements. (D) GroEL/ES-mediated refolding of recombinant WT and A77C DapA.
Fig. 3.
Fig. 3.
Analysis of synthetic unfolded l- and d-DapA. (A) Analytical RP-HPLC of recombinant (black), synthetic l- (red), and synthetic d-DapA (blue) on C4 column (linear gradient 5–100% buffer B over 30 min; buffer A, 0.1% TFA in water; buffer B, 0.1% TFA in 10% water/90% acetonitrile). (B and C) LC-MS analysis of the synthetic l- and d-DapA, respectively. Observed masses were calculated using the Bayesian Protein Reconstruct tool in Analyst 1.5.1 software (AB Sciex) over the charge states covering 650–1,050 Da. See SI Appendix for larger, detailed mass spectra of the final synthetic products and HPLC and LC-MS characterization of all synthetic intermediates.
Fig. 4.
Fig. 4.
Structural and functional characterization of synthetic folded l- and d-DapA. (A) Circular dichroism spectra of Arg-folded and SEC-purified recombinant (black), synthetic l- (red), and synthetic d-DapA (blue). (B) Enzyme activity of Arg-folded and SEC-purified synthetic l- and synthetic d-DapA compared with native recombinant DapA. Error bars indicate SD of at least three assays.
Fig. 5.
Fig. 5.
GroEL/ES-mediated refolding of synthetic l- and d-DapA. Refolding of recombinant (black), synthetic l- (red), and synthetic d-DapA (blue) (250 nM) in the presence (closed circles) or absence (open circles) of 7 μM GroEL/ES. Data are normalized to the maximum point in the GroEL/ES refolding of recombinant DapA.
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